Nucleic acid enrichment

ABSTRACT

This invention relates to methods, reagents and kits for enriching nucleic acid sequences. More particularly, the present invention relates to methods, reagents and kits for sample preparation including sample modification, sample enrichment and amplification

FIELD OF THE INVENTION

This invention relates to methods, reagents and kits for enrichingnucleic acid sequences. More particularly, the present invention relatesto methods, reagents and kits for sample preparation including samplemodification, sample enrichment and amplification.

BACKGROUND

Haplotype information can be vital in the analysis of disease bydetermining whether two or more sequence variants are located on thesame nucleic acid fragment. This is of special interest in tumourresearch and diagnosis where it is important to know if two or moreinactivating mutations occur on the same or different chromosomes.Similarly better information about which genotypes are located on thesame nucleic acid segment can greatly increase the information derivedfrom genotyping data and in the statistical analysis of genetic linkageor linkage disequilibrium of inherited traits and markers such as singlenucleotide polymorphisms, SNPs (e.g. [1-3]).

To date there has been no method developed that satisfactorily solvesthe problem of how to obtain haplotype information in vitro, that is todetermine which gene variants are located, over some distance, on thesame nucleic acid molecule.

Current analysis of heritable diseases is hampered by the fact thatgiven the genotypes of the parents, it is still often impossible toconfirm if a particular allele is obtained from the mother or from thefather. In theory, with the ability to distinguish between haplotypes,more meioses would be informative and thereby facilitate genetic linkageanalysis. Another area where haplotyping has proven to be of interest isin the study of genetic effects on a subject's response to differentdrugs. Recent publications have shown that haplotype information isimportant to be able to relate genetic factors to a patient's responseto various drugs, [4].

Currently, there are only a few methods available for obtaininghaplotype information. When lineage data and nucleic acid samples areavailable linkage analysis is applicable. It is also possible to usestatistical methods to calculate possible allele-combinations fromallele frequencies to gain information on a haplotype. However, thistechnique can only be used with a small number of alleles at the sametime and on population-size data, and the analysis only providesstatistical evidence for the presence of a given haplotype. Haplotypeinformation can also be gained from hemizygous X- and Y-chromosomes,where haplotypes are immediately apparent from the genotype.

The possibility to study cells with only one autosome chromosome isutilised in some in vivo techniques. One approach is the creation ofrodent-human hybrid cells, for example using the so called “Conversiontechnology” [2].Some of the rodent-human hybrid cells will contain oneof the two possible copies from a human chromosome. A second approach isto use hydatidiform moles, i.e. tissues that due to a fertilisationdefect only contain genetic material from the sperm (completehydatidiform mole) thereby containing only one copy of each chromosome.

There are also some in vitro molecular techniques that can be used todetermine haplotypes. One technique is the sub-cloning of all nucleicacid sequences of interest, isolating individual clones and subsequentlygenotyping them. Allele-specific analysis through Fibre Fluorescent InSitu Hybridisation is another possible approach, however it has not yetbeen convincingly shown to be useful for SNP based haplotyping. A thirdapproach is double PCR Allele Specific Amplification (double-PASA [5], adouble allele-specific Polymerase Chain Reaction (PCR) which giveslinkage information of two adjacent polymorphic sites. Pyrosequencing[6] and mass spectrometry may be used to analyse haplotypes over shortdistances, i.e. <100 nt.

SUMMARY OF THE INVENTION

Methods, reagents and kits to analyse haplotypes, genotypes andenrichment of selected sequences are described herein. These methods andreagents are, in addition to aspects mentioned in the backgroundchapter, also of interest for population genetics, identification oflineage in plant and animal breeding and in analysis of microorganisms.

In one aspect of the invention, a general technique is provided toobtain haplotypes through enrichment for one nucleic acid segment toinclude a specific variant at a given position. Thereby any variantposition in a sample could be used for selection, followed by analysinggenetic variants elsewhere in the same nucleic acid fragment.

In another aspect of the invention the same principle can be used forgenotyping or to generate probes that reveal the genotype at particularloci.

Accordingly, the present invention provides a method for samplepreparation that optionally includes the steps of: (a) cleavage of anucleic acid so that a fragment containing the sequence to beinvestigated is created with or without addition of oligonucleotideprobes (b) selective modification of one variant of the nucleic acidsequences (c) enrichment of the selected variant, and (d) analysis ofthe nucleic acid.

The present invention also provides one or several probes for use in thedescribed methods. A first set of probes/probe preferably directs sitespecific cleavage at predetermined sites of the sample uponhybridisation. Preferably, A second set of probes/probe is used tospecifically modify the sample based upon the presence or absence of agiven sequence variant. A third set of probes is used for amplificationof the sample and a fourth set of probes is used for scoring thegenotypes.

The present invention describes several ways to enrich a nucleic acidsequence or sequences from a multitude of sequences on the basis of thesequence or on the basis of a particular sequence variation at a givenposition.

DETAILED DESCRIPTION OF THE INVENTION

The terms “nucleic acid”, “nucleic acid sequence”, “nucleic acidfragment”, “nucleic acid segment”, “nucleic acid probe”,“oligonucleotide”, “target nucleic acid sequence” or “target sequence”describe interchangeably and without preference, a plurality ofnucleotides, covalently linked as such to form linear molecules of DNAor RNA.

The term “variant” describes interchangeably and without preference anucleic acid encoding a variant, which may for example be selected fromthe group including any one or more of the following; a singlenucleotide sequence variant, deletion sequence variant, insertionsequence variant, sequence length variants, and sequence variation amongparalogous or orthologous nucleic acid sequence, or among editedsequences or splice variants

Examples of different approaches are as follows.

The first approach, described in part in FIG. 1, is based on cleavage ofDNA at any predetermined site through the use of so called nucleic acidadapters, hereafter called adapters, that are targets or part of targetsfor restriction enzymes preferably type II or type IIs restrictionenzymes [7,8]. Adapters and sample are mixed, denatured and subsequentlyallowed to cool. The adapters hybridise to their complementary regionsin the sample nucleic acid. One of the adapters is positioned so thatthe resulting cleaved sample DNA contains a variant position at the 5′position (A). Added restriction enzymes cleave the sample and, throughaddition of a ligation template that anneals to both the 5′ and 3′ endof the cleaved sample DNA, circular molecules are obtained by ligationof the ends that are brought next to each other (B). Thiscircularisation is driven by the higher relative concentration of twoends belonging to the same molecule compared to those of two differentcopies of the same or similar molecules. If the added template iscomplementary to the sample DNA-ends, juxtaposing these, then ligationof the two ends can occur. If a mismatch between the sample DNA and theligation template exists at the variant position used for selection, orif there are no free ends at the site intended for ligation, thenligation will not occur. Circularised molecules can then be enriched forthrough the use of exonucleases that degrade uncircularised DNA, and/oramplification of the circularised DNA, for example with rolling circleamplification (RCA) can be performed ([9,10]).

Alternatively, the adapter could be positioned upstream of the variantposition used for selection. Optionally this adapter could be completelyomitted. After cleavage, as described earlier, one or a plurality ofoligonucleotides is added, (template), which hybridises to both the 3′end and to an upstream sequence around the variant position, as shown inFIG. 2A. This provides a specificity step. The structure is then cleavedby chemical, enzyme or other means to generate a structure, as shown inFIG. 2B. Where an enzyme is used, any enzyme capable of cleaving such astructure may be used [11]. The enzyme is preferably selected from, FENnuclease, Mja nuclease, native or recombinant polymerase from Thermusaquatiqus, Thermus thermophilus, or Thermus flavus, or any enzymeselected according to the teachings of Lyamichev et al [11] or U.S. Pat.No. 5,846,717, which are incorporated herein by reference. The variantposition used for selection can either be removed by cleavage, or thecleavage can be performed so that the variant position is the 5′-mostnucleotide of the sequence. Hence the major selective step is in thesubsequent ligation reaction. The use of nucleic acid ligation forallele distinction is well described in the literature, for example[12,13]. To ensure that the cleaved substrate is eligible for ligationthe 3′ sample nucleotide must be complementary to the added template.This can be achieved directly from cleavage of the sample, in which caseit is possible to ligate the DNA directly.

Another approach, which confers increased specificity, is to constructthe added template so that it contains one extra nucleotide, giving agap between the hybridised 3′ and 5′ sequences, similar to that observedfor the SNP. By adding only the complementary nucleotide to the cleavagereaction a substrate for cleavage will only be generated from nucleicacid sequences that contain the complementary nucleic acid sequence.

Yet another approach is to construct the added template so that therewill be a gap. This gap may be filled in by the addition of acomplementary oligonucleotide, as shown in FIG. 2C. Optionally, this gapfilling oligonucleotide can be labelled with an affinity tag, forexample a specific sequence or specific molecule for subsequent affinitypurification. The gap filling oligonucleotide can also be of a specificsequence to be used for circular DNA amplification as described inco-pending application PCT/SE02/01378.

Cleavage of the sample DNA can also be achieved with restriction enzymesthrough the addition of oligonucleotides that hybridise to the selectedsequence. The 5′ cleavage site may or may not be influenced by thevariable sequence. Circularisation and selection is then conducted viaany of the above-mentioned approaches.

Instead of circularising the DNA, the nucleic acid fragment ends can beprotected via addition of protecting adapters to one or both ends basedon selective addition at a variant position at at least one of the ends,as shown in FIG. 3. Generation of the 3′ or 5′ sample ends could beachieved either through cleavage at the variable position or upstream ata generic site, as previously described. In the latter case cleavagewill be performed via structure-specific cleavage as previouslydescribed. This protected linear substrate can now be enriched for,through degradation of unprotected sample using exonucleases. Selectiveamplification of the protected allele can be performed based on thepresence of the added sequence/sequences.

It is not necessary to generate restriction sites in the sample or todenature double stranded DNA. Any number of restriction enzymes havingrecognition sequences located on either side but not within the sequenceof interest, can be used.

Double stranded DNA can be digested at a multitude of sites with one orseveral different restriction enzymes. Digestion at one or several ofthe sites may or may not be affected by a sequence variant. If onespecific sequence variant affects digestion by a restriction enzyme at agiven site, only one of the alleles will become circularised uponligation with a ligation template in the form a ligation casette. Aligation cassette consists of a pair of prehybridized complementaryoligonucleotides with single stranded sequences protruding at one orboth ends to form a correct ligation site for the choosen sequences tobe ligated. Only the circularised allele becomes a template for circularamplification by e.g. rolling circle amplification. If the sample iskept double stranded throughout the process the RCA amplified allelewill be the only single stranded DNA in the sample. This single strandedDNA can then be genotpyed by a single strand specific genotyping methodsuch as, including by way of example only, padlock probes,oligonucleotide ligation assay or invader assay. The principle ofspecifically generate only a subset of a sample single stranded can beutlized with any method capable of performing such an action and is notto be limited to the one mentioned. Subsequent analysis with singlestrand specific methods reveals the genotype of only the selected, andthus single stranded sequence.

In one version an exonuclease is added to make one or both ends of arestriction enzyme digested double stranded sample partially singlestranded before circularization. A choosen specific sequence iscircularized, templated by an added oligonucleotide or pairs ofoligonucleotides either directly or via a structure-specific enzyme cut,as described above, followed by specific ligation. The strands are thengap-filled, followed by DNA ligation. Only the correct allele can bemade into a complete circle possible to amplify with RCA.

A further variant to generate single stranded DNA from a restrictionenzyme digested of a double stranded sample is to specifically degradeonly one of the strands with exonucleases. This can be achieved, by wayof example only, through making a proper choice of restriction enzymesthat will produce a sticky end that is not a substrate for the chosenexonuclease or exonucleases; or via DNA ligation adding a protectingsequence to one or both of the ends; or via DNA ligation add achemically modified and protected sequence to one or both of the ends.The single stranded DNA can then be circularized, either directly viaspecific ligation or via a structure-specific enzyme cut followed byspecific ligation as previously described.

It is also possible to circularise double stranded DNA with or withoutthe addition of a ligation cassette, as described earlier, to make oneof the strands so it can prime an RCA with the intact circularisedstrand as template. After a predefined time the polymerase isinactivated and an oligonucleotide complementary to a specific part ofthe amplification product is added so that it creates a restrictionenzyme site in one of the alleles. After restriction enzyme digestionthe digested allele is recircularised, according to the description inco-pending application PCT/SE02/01378, making it resistant toexonuclease degradation. After exonuclease treatment, serving to degradeall linear nucleic acids and thereby avoiding branched amplification ofthe non-circularized allele, a second generation RCA is conducted,primed with a second oligonucleotide, effectively amplifying only thecircularised allele.

The enriched sample can be subjected to genotyping through any methodand compared to results from genotyping of the total sample. Examples ofmethods which may be used are oligonucleotide ligation assays [12],padlock probes [13], primer extension assays [14], pyrosequencing [15],invader technology[16], mass-spectroscopy [17] or homogenous PCR methodse.g. Taqman [18] or molecular beacons [19]. However, other methods maybe employed with equal utility. By using the enriched sample instead ofa whole sample as the test sample it is also feasible to use anysuitable method-, to find new/unknown mutations or polymorphisms.Thereby all possible mutations in the enriched segment may be detected,also unknown ones, for example by Sanger sequencing or by hybridisingthe enriched sample to an array in order to resequence the sample and inthis respect also find new or unknown mutations. The methods could be,but are not limited to the use of, mismatch recognising enzymes forexample T4 endo VII [20], DHPLC resequencing, Sanger or array, orpyrosequencing [15]. However, other methods may be employed with equalutility. The resulting genotypes will reveal the specific haplotype ofthe sample.

Accordingly, the present invention provides one or several sets ofprobes. A first set of probes/probe direct site specific cleavage atpredetermined sites of the sample upon hybridisation. A second set ofprobes/probe is used to specifically modify the sample based upon asequence variant. A third set of probes is used for amplification of thesample and a fourth set of probes is used for scoring the genotypes.

Instead of investigating the genotypes all along the selected nucleicacid one can use the same principle for genotyping the variant positionused for selection. Upon cleavage of sample DNA an oligonucleotide canbe added that anneals to the 3′ end of a generated fragment and to astretch upstream, around the variant position to be scored, so that aprobe with a hybridising region at its 5′ end is formed, (as shown inFIG. 4A), or a probe with a non-hybridising region at its 5′ end isformed, (as shown in FIG. 4B). If necessary this structure can then becleaved as previously described. The use of ligase will complete thenucleic acid circle. The circle can then be enriched for, usingexonuclease treatment and nucleic acid amplification, preferably rollingcircle amplification. Preferentially the oligonucleotide added containsa sequence between the 3′ and the 5′ hybridising end that consist of aselected sequence used for later hybridisation that can be rendereddouble stranded through the addition of a second oligonucleotide, shownin FIG. 4A as object 1. The added oligonucleotide could contain arecognition sequence for a type IIs restriction enzyme and preferably asequence as dissimilar as possible compared to other oligonucleotidesused for other loci, as described in co-pending applicationPCT/SE02/01378, the contents of which are incorporated herein byreference. Detection of the circularised nucleic acid or amplificationproducts templated by the circularised nucleic acid is used to score thegenotype of the selected position.

Due to the intramolecular nature of the ligation reaction it is feasibleto perform many reactions at the same time (from one to several tens ofthousands). At any practical concentration the fragments willcircularise intramolecularly in preference to intermolecular reactions.

Accordingly, the present invention further provides one or a set ofprobes. A first set of probes/probe directs site-specific cleavage atpredetermined sites of the sample upon hybridisation. A second set ofprobes/probe is used to specifically modify the sample based upon asequence variant. A third set of probes is used for amplification of theenriched sample.

The variant position could be, but is not limited to a sequence variantpolymorphism which may be selected from the group including any one ormore; deletion variant, insertion variant, sequence length variant,single nucleotide polymorphism, substitution variant, paralogous ororthologous nucleic acid sequences, edited sequences or splice variants.

The present invention is also to be used as a mean to isolate and enrichfor a specific sequence or sequences among a multitude of sequences,with the intention of further manipulation of the enrichedsequence/sequences. The methods could be any, sole or a combination ofbut not limited to, amplification, quantification, sequencing, variantscoring, using the enriched sequence/sequences as probes or to comparedifferent enriched samples on the basis of for example amount of sample.

Accordingly, the present invention further provides one or a set ofprobes. A first set of probes/probe directs site specific cleavage atpredetermined sites of the sample upon hybridisation. A second set ofprobes/probe is used to specifically modify the sample based upon anucleotide sequence. A third set of probes is used for amplification ofthe enriched sample.

In all of the above-mentioned methods where DNA samples are mentionedthey could be exchanged with RNA or cDNA samples.

An added oligonucleotide probe can also be treated by the sameprinciples and to be used for subsequent genotyping, as shown in FIG. 5,if the added oligonucleotide anneals forming a non-hybridising region atthe 5′ end. Cleavage of this structure will generate a molecule that canbe circularised with a ligase. Ligation will depend on whether the 5′nucleotide is matched or not with the sample. This circularised probecan then be detected either directly or via the presence ofamplification products (based on the presence of the circle oramplification products of the circle). The presence of such a productdescribes the nature of the variant position. The added oligonucleotidecould preferentially contain a molecule or sequence in the 5′ part thatis used as an affinity tag for removal of unmodified circles beforeamplification of the circularised probes.

Accordingly, the present invention provides one or a set of probes. Afirst set of probes to be specifically modified based on the nature of anucleotide in the target nucleic acid. A second set of probes could beused for purification of the sample. A third set of probes is used foramplification of the modified probes.

Embodiments of the invention will now be described in greater detail, byway of example only not in any way to limit the invention, withreference to the accompanying drawings, of which;

FIG. 1 is a schematic representation of cleavage and circularisation ofsample nucleic acid through the use of adapters;

FIG. 2 is a schematic representation of structure specific cleavage forcircularisation of sample nucleic acids;

FIG. 3 is a schematic representation of addition of protecting ends to alinear nucleic acid sample;

FIG. 4 is a schematic representation of the use of gap-oligonucleotidesfor circularisation of sample nucleic acids;

FIG. 5 is a schematic representation of scoring SNPs throughcircularisation of nucleic acid probes;

FIG. 6 shows (A) the result from a real-time PCR experiment and (B) thegel of the same amplification reactions from an experiment of cleaving,ligating and rolling circle amplification of BAC DNA;

FIG. 7 is a schematic representation (A) of the experimental set-up fordetection of circularisation of nucleic acids via inverse PCR and (B) aphoto of an agarose gel showing the result of such an experiment whereBAC DNA cut with FokI adapters, circularised with ligase, circularmolecules enriched for via exonucleases and finally used for template inan inverse PCR reaction;

FIG. 8 is showing an image of a poly acrylamide gel of radioactivelabelled nucleic acids showing cleavage and ligation of structurespecific cleaved nucleic acids with native DNA Taq polymerase and Tthligase; and

FIG. 9 is showing a photo of an ethidium bromide stained gel ofamplification products obtained from an experiment with cleaved BAC DNAthat had been circularised via cleavage by a structure specific enzymeand the two ends joined by a ligase.

EXAMPLES Example 1

Circularisation of DNA after cleavage with restriction enzymes followedby enrichment through exonuclease treatment and rolling circleamplification. (See FIG. 4)

A BAC clone (RP11-381L18, BacPac resources, Children's hospital,Oakland) with a genomic fragment containing the gene ATP7B was used. DNAwas isolated by the rapid alkaline lysis miniprep method and the DNAconcentration was determined measuring UV A₂₆₀.

HpaII 5 U (New England Biolabs) was used to cleave a double stranded(ds) template in buffer (10 mM Tris-HCl pH 7.5, 10 mM MgCl₂, 1 mM DTT)for 2 hours at 37° C. before heat-inactivation of the enzyme. Two pmolof the ds template was cleaved with HpaII. After the cleavage, thereaction was diluted to different concentrations (10⁴-10⁸ molecules/μl).

The template was ligated into a circle using 0.5 units of T4 DNA ligase,1×T4 DNA ligase bf (66 mM Tris-HCl pH 7.6, 6.6 mM MgCl₂, 10 mM DTT, 66μM ATP) and 10 nM ligation template, 5′Biotin-tt ttt ttt ttt ttt gtc tggaaa gca aac cgg tgc cca ccc atg a 3′ SEQ ID NO1, in each reaction. Afterdenaturation and subsequent addition of ligase to half of the reactions(see below), the samples were incubated at 37° C. for 30 min and thenthe ligase was heat-inactivated at 65° C. for 20 minutes

After ligation, the samples were treated with exonucleases. ExonucleaseV (5 units) was used for 30 min 37° C. before heat-inactivation. Theresult was detected by performing a PCR with the following primers, 5acg ccc acg gct gtc at 3′ SEQ ID NO2 and 5′ tgg acg tct gga aag caa a 3′SEQ ID NO3, (1 μM) located on both sides of the ligation junction. In 50mM Tris HCl pH 8,3, 50 mM KCl, 200 μM dNTP, 0.125 u Taq GOLD polymerase(Perkin Elmer), 0.08×SYBR Green (Molecular Probes) as reporter molecule,and 1xROX (Molecular Probes) as standard, temperature cycles as follows95° C. 10 min activation of Taq polymerase followed by 40 cycles of 95°C. 20 sec, 52° C. 1 min, 72° C. 20 sec. The experiments yield a cyclethreshold value, Ct which is inversely proportional to the amount ofstarting material in the sample.

After the PCR amplification the reactions products were electrophoresedin a 3% agarose gel to ensure that a product of the correct length hadbeen produced.

The results are shown in FIG. 4, where A) Graph showing the fluorescencereadings from a real-time PCR experiment read in an ABI 7700. Thefigures to the left corresponds to the numberings in B. Reactions wereas follows; #2,3—No template control, #4 sample+ligase, #5Sample−ligase, #6 sample+ligase+RCA, #7 Sample—ligase+RCA

B) A 3% agarose gel of the PCR reactions shown in A. Lane 1 in B isloaded with a 100 bp-ladder (lowest band around 50 bp). Lane 2-7corresponds to the same reactions. The arrow denotes the size for acorrect length product.

Example 2

Enrichment of circular DNA over non-circular DNA through the use ofdifferent exonucleases.

BAC DNA as described in example 1 were cleaved and ligated as describedin EXAMPLE 1. Half of the sample was ligated with T4 DNA ligase and halfof the sample was not. The two reactions were further divided into fivedifferent reactions of each (+/−ligase) treated as follows.

-   -   1 5 u ExoV and 1 mM ATP,    -   2. 5 u ExoI, 50 u ExoIII and 25 u T7gene6    -   3. 5 u ExoI 50 u ExoIII and 2,45 u Lambda exo    -   4. 50 u ExoIII, 0,5 u ExoVII and 2,45 u Lambda Exo    -   5. 5 u ExoI, 0,5 u ExoVII in 1×Tris buffer

All reactions were incubated at 37° C. for 30 minutes before heatinactivation of the nucleases at 80° C. for 20 minutes. The results weredetermined as described in example 1. After the PCR amplification, thereaction products were electrophoresed in a 3% agarose gel, and thenucleic acid visualised to ensure that a product of the correct lengthhad been produced (not shown). The results are shown in table 1. TABLE 1Shows the result from an exonuclease treatment of cleave DNA that hadbeen or had not been circularised with ligase. Exo treatment: +ligase:Ct value −ligase: Ct value ExoV(5 U) + ATP(1 mM) 24.49 35.82 ExoI (5U) + ExoIII (50 U) + 21.33 33.52 T7Gen6 (25 U) ExoI (5 U) + ExoIII (50U) + 21.52 35.07 λExo (2.45 U) ExoIII (50 U) + ExoVII 22.85 35.70 (0.5U) + λExo (2.45 U) ExoI (5 U) + ExoVII 28.05 34.70 (0.5 U) + 1× Tris bf

Example 3

Circularisation of DNA after denaturation of dsDNA, hybridisation ofFokI adapters, cleavage of the DNA at predetermined sites, specificcircularisation of the cleaved fragment based on an SNP at the 5′primeend and enrichment of the circularised DNA. (See FIG. 7)

BAC DNA was purified as described in example 1.

BAC DNA was diluted in a series and denatured by heat. Afterdenaturation the samples were directly put on ice.

Different amounts (10¹-10¹⁰ molecules) of BAC DNA were cleaved with 2units FokI and 2 fmol FokI adapters (FokI adapter 5′UTR 5′ cgc atc ccacgt ggg atg cga aag caa aca ggg gt 3′ SEQ ID NO 4, FokI adapter C2930TC-allele 5′ gcc atc cgt gca cgg atg gct gca cag cac cgt gat 3′ SEQ IDNO5, FokI adapter C2930T T-allele 5′ gcc atc cgt gca cgg atg gct gca cagcac cat gat 3′ SEQ ID NO6) in 10 mM Tris-HCl pH 7.5, 10 mM MgCl₂, 1 mMDTT, 50 mM NaCl, 1 xBSA1 for 2 hours 37° C. before heat-inactivation ofthe enzyme.

The ends of the generated fragment nucleic acid were ligated into acircle using 8 fmol of the correct/incorrect ligation template (20+20WDgDNA 5′UTR-Ex13 C-allele, 5′ ctc ggc tct aaa gca aac agg tga tgg acgtct gga aag ctt t 3′ SEQ ID NO7, 20+20 WDgDNA 5′UTR-Ex13 T-allele 5′ ctcggc tct aaa gca aac aga tga tgg acg tct gga aag ctt t 3′ SEQ ID NO8).One unit T4 DNA ligase and 1×T4 DNA ligase buffer was used, and thereactions were incubated for approximately 30 minutes at 37° C. beforeheat-inactivation of the DNA ligase. The circles were exonucleasetreated with 5 units ExoV and 1 mM ATP and the samples were incubated in37° C. for 30 min before heat-inactivation at 80° C. for 20 minutes.

PCR amplification was performed with primers (Frw WDgDNA 5′UTR-Ex13 5′cag agg tga tca tcc ggt ttg 3′ SEQ ID NO9, Rew WDgDNA 5′UTR-Ex13 5′ ggagag gag gcg cag agt gt 3′ SEQ ID NO10), 0.5 μM of each, located on bothsides of the ligation junction. With a total volume of 50 μl, 200 μMdNTP, 1 unit Taq GOLD polymerase, 1×PCR buffer (10 mM Tris-HCl pH 8.3,50 mM KCl, 1.5 mM MgCl₂, 0.001% (w/v) gelatine). 40 amplification cycleswere run after activation of the polymerase: 95° C. 15 sec, 58° C. 1 minand 72° C. 20 sec. The amplified nucleic acids were detected byelectrophoresis in a 3% agarose gel and visualisation by staining withethidium bromide.

The results are shown in FIG. 6B. The following samples were loaded intothe different lanes; 1—Marker, 2 No template control, 3-8 samples from a10-fold dilution series (10¹⁰-10¹) of BAC DNA with correct ligationtemplate and ligase, 9 sample with correct ligation template but minusligase, 10-12 samples from a 10-fold dilution series (10e10 to 10e9)with a ligation template corresponding to the wrong allele, T instead ofC). The arrow denotes the size of a correct length product.

Example 4

Selective ligation of oligonucleotides cleaved with a structure specificenzyme. (See FIG. 8)

The reactions were performed in 1×Tth buffer (1 mM NAD, 10 mM DTT and0,1% Triton X-100). 20 μl reactions containing 0.5 pmol of the upstream,downstream and target oligonucleotides respectively (primer22+1 5′ gtattt gct ggg cac tca ctg ca 3′ SEQ ID NO11, ArmC 5′ tcc aga cgt cca tcacgg tgc tgt gca ttg cct g 3′ SEQ ID NO12 or ArmT 5′ tcc aga cgt cca tcatgg tgc tgt gca ttg cct g 3′ SEQ ID NO13, Template2930 5′cag gca atg cacagc acc gtg cag tga gtg ccc agc aaa tac3′ SEQ ID NO14), 1 unit of Tthligase and native Taq polymerase. The reactions were prepared on ice andinitiated by transfer to a Thermal Cycle where the following program wasrun: 95° C. 20 sec, 72° C. 30 min for 2 cycles. The upstream ordownstream oligonucleotide was radio labelled and the samples wereanalysed on a 10% denaturing polyacrylamide gel. Ten pmol target DNA wasend-labelled with 1.65 pmol γ-³²P dATP (NEN). 4.9 U T4 PNK enzyme and1×T4 PNK buffer (0.05 M Tris-HCl pH 7.6, 10 mM MgCl₂, 10 mM2-mercaptoethanol) was added to each labelling reaction and the tubeswere incubated for 45 min in 37° C. EDTA (1 mM) was added and thesamples were boiled for 5 min in a water bath. The unincorporatednucleotides were removed from the labelling reaction with a MicroSpin™G-50 column (Amersham Pharmacia Biotech).

The experiments with the radio labelled oligonucleotides were detectedon a 10% polyacrylamide gel containing 7 M UREA. The gel was run with0.5×Tris Borat EDTA buffer at 30 W for approximately 30 min and wasdried in a gel dryer for 2 hours 80° C. The dried gel was exposed to aphosphorimager screen overnight.

The results are shown in FIG. 8. Oligonucleotides yielding structure Awas used in experiments 1-6 and oligonucleotides yielding structure Bwas used in experiments 7-12. (i) denotes the size of un-reactedoligonucleotide in experiments 1-6, (ii) the size for ligated product inreactions 1-6, (iii) uncleaved oligonucleotide used in reactions 7-12and (iv) cleaved oligonucleotide in reactions 7-12. 32P denotes aradioactive label on respective oligonucleotide.

Lanes 1-6 shows the results from experiments with oligonucleotide 1labelled with ³²P. Lane 1, T-allel (wrong)—Taq polymerase, lane 2C-allel (correct)—Taq polymerase, lane 3 T-allele—Tth ligase, lane 4C-allel—Tth ligase, lane 5 T-allel, lane 6 C-allele.

Lanes 7-12 show the results from experiments with oligonucleotide 2radio labelled with P³² Lanes 7, 9, 11 is with the T-allele (incorrect)and lane 8,10,12 is with the C-allele (correct). Lanes 7-8 minus Taqpolymerase, lanes 9-10 minus Tth ligase.

Lane 13 shows size markers.

Example 5

Circularisation of BAC DNA after cleavage with restriction enzymes,intramolecular hybridisation and cleavage with a structure specificenzyme followed by ligation, as shown in FIG. 9.

BAC DNA was purified as described in example 1.

Denatured, ss BAC DNA (1×10¹⁰ molecules) was cleaved with 10 unitsDraIII in buffer (10 mM NaCl, 5 mM Tris-HCl, 1 mM MgCl₂, 0.1 mM DTT pH7.9) was used. DraIII was allowed to cleave the DNA for 1 hour 37° C.before heat-inactivation.

This experiment was also done with genomic DNA. 10¹⁰ molecules werecleaved by 1 pmol of each adapter (cleave DraIII up2930 5′ act gga cacaac gtg acg aac ttg ggt 3′ SEQ ID NO15 and cleave DraII down2930 5′ cagggc tca cac gca gtg agt gcc c 3′ SEQ ID NO16) designed to hybridise tosequences in exon 13. The subsequent concerted structure-specificcleavage and ligation reaction contained the same reagents as above and2 pmol of ligation any of two different templates (20+20 DraIII-C₅′ taaacg acc cgt gag tga cgc aca ggt cac ggg ggg ac 3′ SEQ ID NO17 or 20+20DraIII-G 5′ taa acg acc cgt gag tga cgg aca ggt cac ggg ggg ac 3′ SEQ IDNO18). The samples were divided into two parts and on one half wassubjected to a RCA. A real-time PCR was performed on the samples withprimers located on both sides of the ligation junction. In a totalvolume of 50 μl the following reagents were included: 2.5 μl sample,1×PCR bf, 100 μM dNTP, 1 unit Taq GOLD polymerase, 0.5 μM of eachprimer, 1×ROX and 0.08×SYBR. After activating the polymerase 95° C. for10 min, 40 cycles of the following program was run in a Thermal Cycler:95° C. 20 sec, 58° C. 1 min and 72° C. 30 sec.

DraIII and specific adapters designed to hybridise to sequences in exon13 of ATP7B cleaved BAC DNA at predetermined sites. The target DNA wasdenatured to become single-stranded and the adapters were designed tocreate recognition and cleavage sites for DraIII. DraIII cleavagecreated a substrate that was used in the structure-specific cleavage,which generated the 5′ located SNP.

The results are shown in FIG. 8. Shown is the samples run on a 3%agarose gel. Lane 1 is a size marker, lane 2 and 3 PCR no templatecontrols, lane 4 sample, lane 5 without Taq polymerase, lane 6 withoutTth ligase and lane 7 without BAC DNA.

REFERENCES

-   1. Bonnen PE, Story M D, Ashorn C L, Buchholz T A, Weil M M, Nelson    D L: Haplotypes at ATM identify coding-sequence variation and    indicate a region of extensive linkage disequilibrium. Am J Hum    Genet 2000, 67:1437-1451.-   2. Douglas J A, Boehnke M, Gillanders E, Trent J M, Gruber S B:    Experimentally-derived haplotypes substantially increase the    efficiency of linkage disequilibrium studies. Net Genet 2001,    28:361-364.-   3. Stephens J C, Schneider J A, Tanguay D A, Choi J, Acharya T,    Stanley S E, Jiang R, Messer C J, Chew A, Han J H, et al.: Haplotype    variation and linkage disequilibrium in 313 human genes. Science    2001, 293:489-493.-   4. Drysdale C M, McGraw D W, Stack C B, Stephens J C, Judson R S,    Nandabalan K, Arnold K, Ruano G, Liggett S B: Complex promoter and    coding region beta 2-adrenergic receptor haplotypes alter receptor    expression and predict in vivo responsiveness. Proc Natl Acad Sci    USA 2000, 97: 10483-10488.-   5. Liu Q, Thorland E C, Heit J A, Sommer S S: Overlapping PCR for    bidirectional PCR amplification of specific alleles: a rapid    one-tube method for simultaneously differentiating homozygotes and    heterozygotes. Genome Res 1997, 7:389-398.-   6. Ahmadian A, Lundeberg J, Nyren P, Uhlen M, Ronaghi M: Analysis of    the p53 tumor suppressor gene by pyrosequencing. Biotechniques 2000,    28: 140-144, 146-147.-   7. Kim S C, Skowron PM, Szybalski W: Structural requirements for    FokI-DNA interaction and oligodeoxyribonucleotide-instructed    cleavage. J Mol Biol 1996, 258:638-649.-   8. Podhajska A J, Szybalski W: Conversion of the FokI endonuclease    to a universal restriction enzyme: cleavage of phage M13 mp7 DNA at    predetermined sites. Gene 1985, 40:175-182.-   9. Kool E T: Circular oligonucleotides: new concepts in    oligonucleotide design. Annu Rev Biophys Biomol Struct 1996,    25:1-28.-   10. Baner J, Nilsson M, Mendel-Hartvig M, Landegren U: Signal    amplification of padlock probes by rolling circle replication.    Nucleic Acids Res 1998, 26:5073-5078.-   11. Lyamichev V, Brow M A, Dahlberg J E: Structure-specific    endonucleolytic cleavage of nucleic acids by eubacterial DNA    polymerases. Science 1993, 260:778-783.-   12. Landegren U, Kaiser R, Sanders J, Hood L: A ligase-mediated gene    detection technique. Science 1988, 241.-   13. Nilsson M, Malmgren H, Samiotaki M, Kwiatkowski M, Chowdhary B    P, Landegren U: Padlock probes: circularizing oligonucleotides for    localized DNA detection. Science 1994, 265:2085-2088.-   14. Syvanen A C, Aalto-Setala K, Harju L, Kontula K, Soderlund H: A    primer-guided nucleotide incorporation assay in the genotyping of    apolipoprotein E. Genomics 1990, 8:684-692.-   15. Ronaghi M, Karamohamed S, Pettersson B. Uhlen M, Nyren P:    Real-time DNA sequencing using detection of pyrophosphate release.    Anal Biochem 1996, 242:84-89.-   16. Lyamichev V, Mast A L, Hall J G, Prudent J R, Kaiser M W, Takova    T, Kwiatkowski R W, Sander T J, de Arruda M, Arco DA, et al.:    Polymorphism identification and quantitative detection of genomic    DNA by invasive cleavage of oligonucleotide probes. Nat Biotechnol    1999, 17:292-296.-   17. Griffin T J, Hall J G, Prudent J R, Smith L M: Direct genetic    analysis by matrix-assisted laser desorptionlionization mass    spectrometry. Proc Natl Acad Sci USA 1999, 96:6301-6306.-   18. Heid C A, Stevens J, Livak K J, Williams P M: Real time    quantitative PCR. Genome Res 1996, 6:986-994.-   19. Tyagi S, Kramer F R: Molecular beacons: probes that fluoresce    upon hybridization. Nat Biotechnol 1996, 14:303-308.-   20. Mashal R D, Koontz J, Sklar J: Detection of mutations by    cleavage of DNA heteroduplexes with bacteriophage resolvases. Nat    Genet 1995, 9:177-183.

1. of enriching a preselected nucleic acid segment from a mixture ofnucleic acid sequences, the preselected nucleic acid sequenceencompassing a specific variant at a given position, the methodcomprising the steps of: (a) providing a nucleic acid mixture ofsequences which includes the preselected nucleic acid segment to beenriched; (b) cleaving the nucleic acid sequences in the mixture toprovide a nucleic acid fragment comprising the preselected nucleic acidsegment; (c) providing a template oligonucleotide, one end of whichhybridises to a sequence of the segment at or close to the variantposition, and the other end of which hybridises to the end of aprotecting sequence; (d) hybridising the template to the nucleic acidsegment and to the protecting sequence such that the variant positionand the end of the protecting sequence are brought into proximity ofeach other; (e) joining the end of the protecting sequence to thenucleic acid segment to form a ligated product, which ligated product isprotected from degradation; and (f) enriching for the ligated sequence.2. A method according to claim 1, step (b), in which cleavage generatesa variant position located either at or close to one of the ends.
 3. Amethod according to claim 1, step (f), in which the un-ligated productis degraded.
 4. A method according to claim 1, step (f) in which theligated product is amplified.
 5. A method according to claim 1, step(f), in which un-ligated product is degraded and ligated product isamplified.
 6. A method according to claim 1, step (b), in which thevariant position is located at one end of the nucleic acid sequence anda defined nucleic acid sequence is located at the opposite end.
 7. Amethod according to claim 1, in which the cleavage generates a variantposition located at or downstream of the 5′ end of the segment.
 8. Amethod according to claim 3, in which degradation is effected with anexonuclease enzyme.
 9. A method as claimed in claim 1, in which thehybridisation of the template generates a structure that is a substratefor cleavage by a structure specific enzyme.
 10. A method according toclaim 9, in which the structure specific enzyme is selected from thegroup including any one or more; Native or recombinant Fen nuclease,Native or recombinant Mja nuclease Recombinant polymerase from Thermusaquatiqus, Native or recombinant polymerase from Thermus thermophilus,and Native or recombinant polymerase from Thermus flavus.
 11. A methodaccording to claim 9, in which the structure specific enzyme is nativepolymerase from Thermus aquaticus.
 12. A method according to claim 1, inwhich the protecting sequence is the opposite end of the nucleic acidsegment, and in which the ligating step (1 e) circularises the nucleicacid segment.
 13. A method according to claim 1, in which the nucleicacid segment comprises DNA.
 14. A method according to claim 1, in whichthe nucleic acid segment comprises RNA.
 15. A method according to claim1, in which the amplifying step comprises rolling circle amplification.16. A method according to claim 1, in which the protecting sequence is anucleic acid adapter with a protected end.
 17. A method according toclaim 1, in which, cleavage of the nucleic acid sample in step (lb) usesat least one adapter and at least one restriction enzyme.
 18. A methodaccording to claim 17 in which, the one or more adapters hybridises tothe nucleic acid sample such that, upon cleavage, the variant positionis located at one end of the segment.
 19. A method according to claim 1,in which there is a gap between the hybridised 3′ end and the hybridisedupstream sequence of the nucleic acid segment.
 20. A method according toclaim 19 in which, one of the sequences is extended to fill the gapthere between.
 21. A method according to claim 19, in which one of thesequences is extended with a polymerase.
 22. A method according to claim19, in which the sequence is extended with an enzyme selected from thegroup including any or more; Native or recombinant polymerase fromThermus aquatiqus, Native or recombinant polymerase from Thermusthermophilus, and Native or recombinant polymerase from Thermus flavus.23. A method according to claim 19, in which one of the sequences isextended with Klenow.
 24. A method according to claim 19 in which, thegap is closed in with an oligonucleotide.
 25. A method according toclaim 24, in which the oligonucleotide inserted into the gap is labelledwith an affinity tag.
 26. A method according to claim 24, in which theoligonucleotide is labelled with biotin.
 27. A method as claimed inclaim 1, which includes an initial step of denaturing the nucleic acidwhen the nucleic acid is at least partially double stranded.
 28. Amethod of analysing a target nucleic acid sequence for the presence of amutation at a given variant position comprising the steps of: a)providing a probe for the target sequence, the probe having a 3′ endwhich is complementary to part of the target sequence, and having asequence downstream of the 5′ end which is complementary to an adjacentpart of the target sequence when the target sequence includes themutation; b) hybridising the probe with the target sequence, forming anon-hybridising region at the 5′ end of the probe; c) cleavage of thenon hybridising region of the probe by a structure specific enzyme; d)circularisation of the probe; e) removal of uncircularised probe; f)optionally amplifying the circularised probe; g) and detection of theamplified product.
 29. A method according to claim 28, in which thestructure specific enzyme is selected from the group including any oneor more; Native or recombinant Fen nuclease, Native or recombinant Mjanuclease Recombinant polymerase from Thermus aquatiqus, Native orrecombinant polymerase from Thermus thermophilus, and Native orrecombinant polymerase from Thermus flavus.
 30. A method according toclaim 28, in which the structure specific enzyme is native Taqpolymerase.
 31. A method according to claim 28, in which the removalofuncircularised probes in step (28 e) comprises exonuclease treatment.32. A method according to any claim 28, in which the removalofuncircularised probes (28e) is by binding to a portion in the 5′ endof the probe that is cleaved off if hybridised to the sample.
 33. Amethod according to claim 28, in which the amplification step (28 f)comprises rolling circle amplification.
 34. A method according claim 28,in which the detection of the product, step (28 g) is by detection ofthe circularised probe directly or by detection of amplificationproducts thereof.
 35. A method according to claim 28, in which a gap ispresent between the hybridised 3′ end and the hybridised upstreamsequence.
 36. A method according to claim 28, in which one of thesequences is extended to fill the gap therebetween.
 37. A methodaccording to claim 35, in which one of the sequences is extended with apolymerase.
 38. A method according to claim 36, in which the sequence isextended with an enzyme selected from the group including any one ormore; Recombinant polymerase from Thermus aquatiqus, Native orrecombinant polymerase from Thermus thermophilus, and Native orrecombinant polymerase from Thermus flavus.
 39. A method according toclaim 36 in which one of the sequences is extended with nativepolymerase from Thermus aquaticus.
 40. A method according to claim 36,in which one of the sequences is extended with Klenow.
 41. A methodaccording to claim 35 in which, the gap is closed in with anoligonucleotide.
 42. A method according to claim 35 in which theoligonucleotide inserted into the gap is labelled with an affinity tag.43. A method according to claim 28, in which the oligonucleotide islabelled with biotin.
 44. A method according to claim 28 in which theprobe is labelled with a detectable cleavable molecule.
 45. A methodaccording to claim 44 in which, the detectable cleavable molecule isfluorescent.
 46. A method according to claim 44, in which cleavage ofthe probe is detected by fluorescence.
 47. A method according to claim1, in which the specific variant is a polymorphism, wherein the processincludes a further step of variant scorng.
 48. A method according toclaim 1, in which the specific variant is a single nucleotide variant,wherein the process includes a further step of single nucleotide variantscoring.
 49. A method according to claim 1, in which the specificvariant is a deletion variant, wherein the process includes a furtherstep of deletion variant scoring.
 50. A method according to claim 1, inwhich the specific variant is an insertion variant wherein the processincludes a further step of insertion variant scoring.
 51. A methodaccording to claim 1, in which the specific variant is a sequencevariation wherein the process includes a further step of sequencevariation scoring.
 52. A method according to claim 1, in which thespecific variant is a sequence length variation, wherein the processincludes a further step of sequence length variation scoring.
 53. Amethod of enriching a preselected nucleic acid segment from a mixture ofnucleic acid sequences, the method comprising the steps of: (a)providing a nucleic acid mixture of sequences which includes thepreselected nucleic acid segment to be enriched; (b) cleaving thenucleic acid sequences in the mixture to provide a nucleic acid fragmentcomprising the preselected nucleic acid segment; (c) providing atemplate oligonucleotide, one end of which hybridises to a sequence ofthe segment, and the other end of which hybridises to the end of aprotecting sequence; (d) hybridising the template to the nucleic acidsegment and to the protecting sequence such that the nucleic acidsegment and the protecting sequence are brought into proximity of eachother; (e) joining the end of the protecting sequence to the nucleicacid segment to form a ligated product, which ligated product isprotected from degradation; and (f) enriching for the ligated sequence.54. A method according to claim 53, step (f), in which the un-ligatedproduct is degraded.
 55. A method according to claim 53, step (f) inwhich the ligated product is amplified.
 56. A method according to claim53, step (f), in which un-ligated product is degraded and ligatedproduct is amplified.
 57. A method according to claim 53, step (b), inwhich a defined nucleic acid sequence is located at least at one of theends.
 58. A method according to claim 53, in which degradation of steps(e) and (f) is effected with an exonuclease enzyme.
 59. A method asclaimed in claim 53, in which the hybridisation of the templategenerates a structure that is a substrate for cleavage by a structurespecific enzyme.
 60. A method according to claim 59, in which thestructure specific enzyme is selected from the group including any oneor more; Native or recombinant Fen nuclease, Native or recombinant Mjanuclease Recombinant polymerase from Thermus aquatiqus, Native orrecombinant polymerase from Thermus thermophilus, and Native orrecombinant polymerase from Thermus flavus.
 61. A method according toclaim 59, in which the structure specific enzyme is native polymerasefrom Thermus aquaticus.
 62. A method according to claim 1, in which theprotecting sequence is the opposite end of the nucleic acid segment, andin which the ligating step (1 e) circularises the nucleic acid segment.63. A method according to claim 1, in which the nucleic acid segmentcomprises DNA.
 64. A method according to claim 1, in which the nucleicacid segment comprises RNA.
 65. A method according to claim 1, in whichthe amplifying step comprises rolling circle amplification.
 66. A methodaccording to claim 55, in which amplification is performed with nativeor recombinant polymerase of phage phi29 or related polymerases.
 67. Amethod according to claim 1, in which the protecting sequence is anucleic acid adapter with a protected end.
 68. A method according toclaim 1, in which, cleavage of the nucleic acid sample in step (53 b)uses at least one adapter and at least one restriction enzyme.
 69. Amethod according to claim 67 in which, the one or more adaptershybridises to the nucleic acid sample such that, upon cleavage, thevariant position is located at one end of the segment.
 70. A methodaccording to claim 1, in which there is a gap between the hybridised 3′end and the hybridised upstream sequence of the nucleic acid segment.71. A method according to claim 69 in which, one of the sequences isextended to fill the gap there between.
 72. A method according to claim69, in which one of the sequences is extended with a polymerase.
 73. Amethod according to claim 70, in which the sequence is extended with anenzyme selected from the group including any or more; Native orrecombinant polymerase from Thermus aquatiqus, Native or recombinantpolymerase from Thermus thermophilus, and Native or recombinantpolymerase from Thermus flavus.
 74. A method according to claim 70, inwhich one of the sequences is extended with Klenow.
 75. A methodaccording to claim 70 in which, the gap is closed in with anoligonucleotide.
 76. A method according to claim 75, in which theoligonucleotide inserted into the gap is labelled with an affinity tag.77. A method according to claim 75, in which the oligonucleotide islabelled with biotin.
 78. A method as claimed in claim 1, which includesan initial step of denaturing the nucleic acid when the nucleic acid isat least partially double stranded.
 79. A method according to claim 55,in which the amplification product is labelled during amplification. 80.A method as claimed in claim 53, in which the selected segment issubjected to a further step of nucleotide sequencing.
 81. A method asclaimed in claim 53, in which the specific segment is subjected to afurther step of single nucleotide variant scoring.
 82. A method asclaimed in claim 53, in which the specific segment is subjected to afurther step of deletion variant scoring.
 83. A method as claimed inclaim 53, in which the specific segment is subjected to a further stepof insertion variant scoring.
 84. A method as claimed in claim 53, inwhich the specific segment is subjected to a further step of sequencevariation scoring.
 85. A method as claimed in claim 53, in which thespecific segment is subjected to a further step of sequence lengthvariation scoring.
 86. A method as claimed in claim 53, in which thespecific segment is subjected to a further step of use as hybridisationprobe for in situ analysis.
 87. A method as claimed in claim 53, inwhich the specific segment is subjected to a further step ofhybridisation to an array.
 88. A method as claimed in claim 53, in whichthe specific segment is subjected to a further step of quantification.